Structural basis for relief of phospholamban-mediated inhibition of the sarcoplasmic reticulum Ca²⁺-ATPase at saturating Ca²⁺ conditions

Abstract

Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) is critical for cardiac Ca²⁺ transport. Reversal of phospholamban (PLB)-mediated SERCA inhibition by saturating Ca²⁺ conditions operates as a physiological rheostat to reactivate SERCA function in the absence of PLB phosphorylation. Here, we performed extensive atomistic molecular dynamics simulations to probe the structural mechanism of this process. Simulation of the inhibitory complex at superphysiological Ca²⁺ concentrations ([Ca²⁺] = 10 mm) revealed that Ca²⁺ ions interact primarily with SERCA and the lipid headgroups, but not with PLB's cytosolic domain or the cytosolic side of the SERCA–PLB interface. At this [Ca²⁺], a single Ca²⁺ ion was translocated from the cytosol to the transmembrane transport sites. We used this Ca²⁺-bound complex as an initial structure to simulate the effects of saturating Ca²⁺ at physiological conditions ([Ca²⁺]_total ≈ 400 μm). At these conditions, ∼30% of the Ca²⁺-bound complexes exhibited structural features consistent with an inhibited state. However, in ∼70% of the Ca²⁺-bound complexes, Ca²⁺ moved to transport site I, recruited Glu⁷⁷¹ and Asp⁸⁰⁰, and disrupted key inhibitory contacts involving the conserved PLB residue Asn³⁴. Structural analysis showed that Ca²⁺ induces only local changes in interresidue inhibitory interactions, but does not induce repositioning or changes in PLB structural dynamics. Upon relief of SERCA inhibition, Ca²⁺ binding produced a site I configuration sufficient for subsequent SERCA activation. We propose that at saturating [Ca²⁺] and in the absence of PLB phosphorylation, binding of a single Ca²⁺ ion in the transport sites rapidly shifts the equilibrium toward a noninhibited SERCA–PLB complex.

Introduction

The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA)² uses the energy derived from hydrolysis of one ATP to transport two Ca²⁺ ions from the cytosol into the lumen of the sarcoplasmic reticulum (SR) (1). In cardiac muscle cells, SERCA function is reversibly regulated by the transmembrane phospholamban (PLB). PLB binds SERCA in a 1:1 heterodimeric regulatory complex and inhibits SERCA activity (2, 3). Phosphorylation of PLB then relieves SERCA inhibition (3) to increase the rate of cardiac muscle relaxation and to restore the SR Ca²⁺ load necessary for muscle contraction in subsequent beats (4).

PLB inhibits SERCA by binding to a large pocket located in the transmembrane (TM) domain of the pump (5–10). Spectroscopy studies have shown that in the bound complex, SERCA inhibition is tightly coupled to a structural transition between inhibitory and noninhibitory structural states of PLB (11–14). More recently, X-ray crystallography studies showed that in its unphosphorylated form, PLB forms specific intermolecular interactions between conserved residue Asn³⁴ and residue Gly⁸⁰¹ of SERCA (15). Extensive studies by our group showed that these intermolecular interactions induce a substantial structural rearrangement of the transmembrane transport sites and stabilize a metal ion-free E1 intermediate of the pump protonated at residue Glu⁷⁷¹, E1·H⁺₇₇₁ (16, 17). This SERCA intermediate serves as a kinetic trap that decreases SERCA's apparent affinity for calcium at low Ca²⁺ concentrations and depresses the structural transitions necessary for Ca²⁺-dependent activation of SERCA (16, 17).

In the absence of PLB phosphorylation, relief of SERCA inhibition occurs at saturating Ca²⁺ concentrations (18, 19). Two mechanisms have been proposed for the relief of SERCA inhibition at saturating Ca²⁺ conditions: the dissociation model and the subunit model. The dissociation model proposes that PLB must physically separate from SERCA to relieve inhibition, whereas the subunit model hypothesizes that PLB acts as a functional subunit of SERCA, and inhibition is relieved by local structural rearrangements within the SERCA–PLB complex. Whereas cross-linking experiments have suggested that saturation Ca²⁺ conditions induce PLB dissociation from SERCA, the findings from these studies are actually consistent with a structural rearrangement, and not dissociation, of the inhibitory complex (7, 19). Furthermore, extensive spectroscopic experiments in live cells, ER membranes, and reconstituted vesicles unequivocally support the subunit model, as they provide direct detection of the SERCA–PLB interaction at high Ca²⁺ concentrations (11, 20–22).

These studies have shown that saturating Ca²⁺ conditions do not induce dissociation of the SERCA–PLB complex, but the spatial and temporal mechanisms by which Ca²⁺ reverses SERCA remain unknown. In this study, we designed a series of atomistic molecular simulations to determine the mechanisms for Ca²⁺-dependent relief of inhibitory interactions in the SERCA–PLB complex. First, we used a full-length structure of SERCA bound to the inhibitory structure of unphosphorylated PLB as a starting structure to obtain a structure of the SERCA–PLB complex at saturating Ca²⁺ conditions. We then used the Ca²⁺-bound SERCA–PLB structural model generated through this computational approach to perform six independent 1-μs molecular dynamics (MD) simulations of the complex at physiologically relevant conditions. This set of independent simulations was used to systematically identify the effects of saturating Ca²⁺ conditions on the inhibitory contacts between SERCA and PLB in the absence of PLB phosphorylation.

Results

Interaction of Ca²⁺ with the SERCA–PLB complex at saturating Ca²⁺ concentrations

Physiologically relevant saturating free Ca²⁺ concentrations are in the low micromolar range (23), but these ion concentrations cannot be effectively modeled with our explicit MD simulations because the finite size of the systems would require less than one Ca²⁺ ion/system. To overcome this limitation, we first performed a 0.5-μs MD simulation of the SERCA–PLB complex at 10 mm CaCl₂ to mimic saturating Ca²⁺ concentrations and to match the experimental conditions used previously in X-ray crystallography studies (24, 25). At this Ca²⁺ concentration, Ca²⁺ ions interact with several acidic residues of SERCA exposed on both sides of the lipid bilayer, as well as with the lipid headgroups (Fig. 1). However, we found no indication that Ca²⁺ interacts with the cytosolic domain of PLB or near the cytosolic side of the SERCA–PLB interface (Fig. 1).

Figure 1.

Map of the Ca²⁺–protein and Ca²⁺–lipid interactions in the MD trajectory of the complex at 10 mm Ca²⁺. The map of the weighted mass density of Ca²⁺ (orange surface representation) was calculated using a grid resolution of 1 Å and a cutoff distance of 3.5 Å between Ca²⁺ and the protein/lipid atoms. The headgroups of the lipid bilayer are shown as gray spheres; SERCA and PLB are shown in blue and red cartoons, respectively. The arrows indicate the location of functionally important sites in SERCA: phosphorylation site (Asp³⁵¹) in green; the K⁺-binding site (Ala⁷¹¹, Ala⁷¹⁴, Lys⁷¹², and Glu⁷³²) in purple; the Nβ5–β6 loop (Asp⁴²⁶–Lys⁴³⁶) in cyan; and the cytosolic gate (Asp⁵⁹ and Glu³⁰⁹) in magenta.

Mapping of Ca²⁺-SERCA interactions revealed that Ca²⁺ ions do not form interactions with functionally important regions of the pump, such as the phosphorylation site (Asp³⁵¹) or the K⁺-binding site involved in SERCA dephosphorylation (Ala⁷¹¹, Ala⁷¹⁴, Lys⁷¹², and Glu⁷³²) (26) (Fig. 1). In other cases, Ca²⁺ ions interact, albeit nonspecifically, with other functional sites in the cytosolic headpiece, such as the Nβ5-β6 loop (Asp⁴²⁶–Lys⁴³⁶) (27) (Fig. 1). We found that Ca²⁺ ions bind to SERCA near the cytosolic gate that leads to the transport sites (Fig. 1). This site is located ∼20 Å away from the SERCA–PLB interface and is not physically altered by PLB, and SERCA–metal ion interactions in this site are completely independent from PLB binding (15, 16). On average, 2–3 Ca²⁺ ions occupy this region of the protein, but only a single Ca²⁺ ion binds to residues Asp⁵⁹ and Glu³⁰⁹ at the entrance of the cytosolic pathway (Fig. 2A). We found that in the submicrosecond timescale (t = 0.34 μs), Glu³⁰⁹ translocates a single Ca²⁺ ion from Asp⁵⁹ to Asp⁸⁰⁰ (Fig. 2B). Ca²⁺ translocation is facilitated by a change in the dihedral angle N-Cα-Cβ-Cγ (χ₁) of Glu³⁰⁹ from values of +165° and −165° to a χ₁ = −65°. Upon translocation, Ca²⁺ is stabilized in the transport sites by electrostatic interactions with residues Asp⁸⁰⁰ and Glu³⁰⁹ (Fig. 2C). This Ca²⁺-bound configuration of the complex is stable for the remainder of the simulation time, so we used it as starting structure for six independent MD simulations, identified as CAL1–CAL6. These MD simulations were used to determine the effects of saturating Ca²⁺ conditions at physiologically relevant conditions (e.g. ∼400 μm total Ca²⁺ and 100 mm K⁺).

Figure 2.

Mechanism for Ca²⁺ binding to the transport sites at superphysiological Ca²⁺ concentrations. A, in the nanosecond timescale, a single Ca²⁺ ion binds to SERCA residues Asn⁵⁹ and Glu³⁰⁹, located at the entrance of the cytosolic pathway leading to the transport sites. B, following Ca²⁺ binding, rotation of the Glu³⁰⁹ side chain facilitates translocation of Ca²⁺ through the cytosolic pathway into the transport sites. C, upon translocation, the position of Ca²⁺ in the transport sites is stabilized by electrostatic interactions with residues Asp⁸⁰⁰ and Glu³⁰⁹ for the remainder of the simulation time. In all panels, the TM helices are represented by gray ribbons, transport site residues are shown as sticks, and the Ca²⁺ ion is represented as a yellow sphere.

This set of MD simulations revealed that Ca²⁺ remains bound to the transport sites of SERCA and does not dissociate back to the cytosol at physiological conditions. We calculated the coordination number and coordination shell of Ca²⁺ to characterize the interactions that stabilize a single Ca²⁺ ion in the transport sites of SERCA. We define the coordination number of Ca²⁺ as the number of oxygen atoms within 3.5 Å of the calcium ion. This distance is normally considered to be the maximum possible distance between ligand oxygen atoms and the calcium ion (28–30). We found that in all trajectories, Ca²⁺ interacts with the transport sites predominantly with a coordination number of 7, although Ca²⁺ also exhibits a coordination number of 8 in a small percentage of the simulation time (<10%). These coordination numbers fall within the typical values estimated from Ca²⁺–protein (31, 32) and Ca²⁺–SERCA complexes (24, 25).

In two trajectories, CAL1 and CAL3, the Ca²⁺ ion interacts predominantly with seven coordinating oxygen atoms primarily in a pentagonal bipyramidal coordination geometry, in agreement with previous crystallographic studies of Ca²⁺–bound SERCA (25) (Fig. 3A). The seven coordinating ligands for Ca²⁺ are the carboxylic oxygen atoms from residues Glu³⁰⁹ and Asp⁸⁰⁰, the carbonyl moiety from residue Asn⁷⁹⁶, and between two and four water molecules (Fig. 3A). We found that in trajectories CAL2 and CAL4–CAL6, Ca²⁺ also coordinates to oxygen atoms within the transport site predominantly in a heptavalent pentagonal bipyramidal geometry. However, Glu⁷⁷¹ rapidly (t = 25–250 ns) replaces Glu³⁰⁹ in the first coordination shell of Ca²⁺ (Fig. 3B). In this binding mode, Ca²⁺ interacts with the oxygen atoms from transport site residues Glu⁷⁷¹ and Asp⁸⁰⁰, the carbonyl group from residue Asn⁷⁹⁶, and 2–3 water molecules (Fig. 3B).

Figure 3.

Structure and ligand coordination of Ca²⁺ in the transport sites of PLB-bound SERCA simulated at physiological conditions. A, representative structures of the first coordination shell of Ca²⁺ found in the trajectories CAL1 and CAL3. Here, Ca²⁺ interacts with Glu³⁰⁹ and Asp⁸⁰⁰ in either monodentate or bidentate coordination geometries; the carboxamide moiety of Asn⁷⁹⁶ and water molecules are also found in the first coordination shell of Ca²⁺. B, structures of the most populated Ca²⁺ coordination geometries found in the trajectories CAL2, CAL4, CAL5, and CAL6. Here, the carboxylic groups of Glu⁷⁷¹ and Asp⁸⁰⁰ act as monodentate (e.g. structures 1 and 2) or bidentate ligands (e.g. structure 3) to bind Ca²⁺ in the transport sites. The coordination geometry of Ca²⁺ in these trajectories also includes Asn⁷⁹⁶ and 2–3 water molecules. In all panels, transport site residues and water molecules are shown as sticks, oxygen atoms in the first coordinating shell of Ca²⁺ are shown as red spheres, and the Ca²⁺ ion is represented as a yellow sphere.

Effect of saturating Ca²⁺ conditions on PLB-induced inhibitory interactions

Recent studies have shown that PLB residue Asn³⁴, which is absolutely required for SERCA inhibition (34), forms specific hydrogen bond interactions with Gly⁸⁰¹ and Thr⁸⁰⁵ in the TM domain of SERCA (15–17). These interactions induce alterations in the transport site geometry that prevent metal ion occlusion in the transport sites (16, 17). The SERCA-Ca²⁺ interactions shown in Fig. 3 suggest that saturating Ca²⁺ concentrations alter SERCA–PLB inhibitory contacts at physiological conditions. Therefore, we measured intermolecular residue pair distances between Gly⁸⁰¹ and Asn³⁴ and between Thr⁸⁰⁵ and Asn³⁴. SERCA residues Glu⁷⁷¹ and Asp⁸⁰⁰ play a key role in Ca²⁺ occlusion in the transport sites (35), so we also measured the interresidue distance between Glu⁷⁷¹ and Asp⁸⁰⁰.

In the trajectories where Ca²⁺ primarily binds to SERCA residues Glu³⁰⁹ and Asp⁸⁰⁰ (trajectories CAL1 and CAL3), PLB residue Asn³⁴ interacts directly with the backbone oxygen of Gly⁸⁰¹ and the side-chain hydroxyl group of Thr⁸⁰⁵ (Fig. 4). In both cases, the intermolecular interactions Asn³⁴–Gly⁸⁰¹ and Asn³⁴–Thr⁸⁰⁵ are present for most of the simulation time. Furthermore, we found that the carboxyl groups of transport site residues Glu⁷⁷¹ and Asp⁸⁰⁰ in these trajectories are separated by a distance of at least 9 Å (Fig. 4). This spatial separation is characteristic of the inhibited SERCA–PLB complex in the absence of Ca²⁺ (15, 16). Therefore, the stability of the inhibitory interactions and the large spatial separation between Glu⁷⁷¹ and Asp⁸⁰⁰ indicate that the structures populated in the trajectories CAL1 and CAL3 correspond to those of an inhibited Ca²⁺-bound state of the SERCA–PLB complex (15–17).

Figure 4.

Distance evolution between residue pairs associated with inhibition Ca²⁺ occlusion calculated from the MD trajectories of the Ca²⁺-bound SERCA–PLB complex. Distances between key inhibitory contacts involving conserved PLB residue Asn³⁴ and SERCA Gly⁸⁰¹ were calculated using atoms N_δ of Asn³⁴ and the backbone oxygen of Gly⁸⁰¹. The distance between Thr⁸⁰⁵ and Asn³⁴ was calculated between the O_γ of Thr⁸⁰⁵ and N_δ of Asn³⁴. Distances between Glu⁷⁷¹ and Asp⁸⁰⁰, which occlude Ca²⁺ in site I, were calculated using atoms C_δ and C_γ, respectively. The dashed lines indicate the interresidue distances in the initial structure-inhibited complex reported in a previous study by our group (16).

In four MD trajectories, CAL2, CAL4, CAL5, and CAL6, we found substantial changes in the distances between intermolecular residue pairs Asn³⁴–Gly⁸⁰¹ and Asn³⁴–Thr⁸⁰⁵. Here, the distance Asn³⁴–Thr⁸⁰⁵ increases by 1–2 Å (Fig. 4); however, the most significant change is the 3-Å increase in the distance between the side chain of Asn³⁴ and the backbone oxygen of Gly⁸⁰¹ (Fig. 4). Most importantly, we found that the spatial separation Asn³⁴ of PLB and Gly⁸⁰¹ of SERCA in these trajectories occurs concomitantly with a 3–4-Å decrease in the distance between transport site residues Glu⁷⁷¹ and Asp⁸⁰⁰ (Fig. 4). These changes in interresidue distances are also accompanied by a shift in the dihedral angle Cα-Cβ-Cγ-Nδ (χ₂) of Asn³⁴ from two narrow distributions at χ_{2 =} +180° and χ₂ = −180° to a single broad distribution with a mean around −20° (Fig. 5). This structural change is mostly characterized by a transition from an extended side-chain conformation to a self-contact (36) involving side-chain nitrogen and backbone oxygen atoms of Asn³⁴ (Fig. 5). These findings indicate that in trajectories CAL2, CAL4, CAL5, and CAL6, the side chain of PLB residue Asn³⁴ becomes more mobile and no longer establishes inhibitory contacts with SERCA.

Figure 5.

χ₂ dihedral angle probability distributions for PLB residue Asn³⁴ in the trajectories of the Ca²⁺-bound SERCA–PLB complex. The insets illustrate both the preferred conformation adopted by Asn³⁴ and the formation or disruption of the inhibitory interaction Asn³⁴–Gly⁸⁰¹ in each individual MD trajectory.

These observations provide evidence that is consistent with relief of SERCA–PLB inhibition at saturating Ca²⁺ conditions. However, this phenomenon is not consistently observed in all six trajectories, which suggests that this disruption of inhibitory contacts probably occurs in equilibrium and in a Ca²⁺-independent manner. To test this hypothesis, we performed six 1-μs MD simulations of the SERCA–PLB complex in the absence of Ca²⁺. We found that whereas intermolecular distance Asn³⁴–Thr⁸⁰⁵ is variable among independent MD trajectories, Asn³⁴ of PLB consistently remains spatially close to SERCA residue Gly⁸⁰¹ throughout the entire simulation time in all trajectories (Fig. 6). We also found that carboxylic groups of transport site residues Glu⁷⁷¹ and Asp⁸⁰⁰ is are separated by a distance larger than 9 Å in all Ca²⁺-free trajectories (Fig. 6); such distance is longer than the 6-Å separation required for Ca²⁺ occlusion in this site (24, 25). Therefore, these findings indicate that relief of SERCA–PLB inhibitory contacts does not occur spontaneously in the absence of Ca²⁺ and that Ca²⁺ binding to SERCA at saturating Ca²⁺ conditions disrupts key SERCA–PLB inhibitory contacts at physiological conditions.

Figure 6.

Distance evolution between residue pairs associated with inhibition Ca²⁺ occlusion calculated from the MD trajectories of the Ca²⁺-free SERCA–PLB complex. Interresidue distances were calculated using the same atom pairs described in the legend to Fig. 4. The dashed lines indicate the distances in the initial structure-inhibited complex reported (16). The plots show that in the absence of Ca²⁺, the inhibitory contacts between Asn³⁴ of PLB and Gly⁸⁰¹ are not disrupted throughout the entire simulation time in all trajectories.

Effects of saturating Ca²⁺ conditions on the structural dynamics of PLB

Our results demonstrate that Ca²⁺ binding to the transport sites of SERCA at saturating Ca²⁺ conditions generally disrupt key inhibitory interactions between SERCA and PLB. However, it is not clear whether disruption of the inhibitory interactions is linked to (i) changes in the structural dynamics of PLB, (ii) a reorganization of the SERCA–PLB interface, or (iii) local changes involving interresidue interactions along the interface. Therefore, we performed extensive measurements of structural parameters to determine the changes in the structural dynamics of PLB in response to saturating Ca²⁺ conditions.

In the both inhibited and noninhibited Ca²⁺-bound complexes, the cytosolic and TM helices that contain the regulatory phosphorylation and inhibitory domains populate an α-helical structure for >95% of the time. Average interhelical angles between the cytosolic (Val⁴–Thr¹⁷) and TM (residues Arg²⁵–Leu⁵²) helices of PLB fluctuate between 52 and 77° (Table 1), which corresponds to a T-shaped architecture of PLB (Fig. 7A). We found that the calculated interhelical angles of PLB in both inhibited and noninhibited complexes are within the range of those determined experimentally for the unphosphorylated PLB monomer in solution (37, 38). These findings are in agreement with previous spectroscopic studies (11, 39) and demonstrate that saturating Ca²⁺ conditions do not induce order-to-disorder transitions associated with PLB phosphorylation (12, 39–43).

Table 1.

Effects of saturating Ca²⁺ conditions on the structural dynamics of PLB and the inhibitory SERCA–PLB contacts

Trajectory	PLB interhelical anglea,b	Change in PLB tilt anglea,c	Fraction of native inhibitory contacts, Qinha,d
			Leu31	Asn34	Phe35	Ile38
	degrees	degrees
CAL1	77.2 ± 8.1	4.5 ± 1.2	0.85 ± 0.06	0.84 ± 0.05	0.86 ± 0.10	0.87 ± 0.05
CAL2	60.0 ± 8.5	3.1 ± 1.2	0.73 ± 0.09	0.55 ± 0.07	0.68 ± 0.10	0.79 ± 0.06
CAL3	51.5 ± 10.5	5.4 ± 2.1	0.81 ± 0.08	0.86 ± 0.06	0.89 ± 0.07	0.84 ± 0.06
CAL4	72.0 ± 7.5	2.5 ± 1.2	0.71 ± 0.09	0.52 ± 0.06	0.71 ± 0.12	0.73 ± 0.09
CAL5	55.1 ± 9.3	3.9 ± 1.5	0.70 ± 0.13	0.62 ± 0.06	0.79 ± 0.06	0.82 ± 0.06
CAL6	56.5 ± 8.7	2.3 ± 1.2	0.73 ± 0.11	0.54 ± 0.05	0.70 ± 0.07	0.73 ± 0.09

^a Values reported as average ± S.D.

^b Interhelical angle is defined as the polar angle between the cytosolic (Val⁴–Thr¹⁷) and TM (residues Arg²⁵–Leu⁵²) helices of PLB.

^c Axial tilt angle was calculated using backbone alignment, with SERCA–PLB crystal structure (PDB entry 4KYT (15)) as a reference.

^d These PLB residues play important roles in inhibition of SERCA (45).

Figure 7.

Effects of saturating Ca²⁺ conditions on the structural dynamics of PLB at physiologically relevant conditions. A, structures of the Ca²⁺-bound SERCA–PLB complex embedded in a lipid bilayer at the end of the each 1-μs MD trajectory. The headgroups of the lipid bilayer are shown as gray spheres; SERCA is shown as a blue surface representation. PLB is shown as a cartoon representation and colored according to its functional domains: cytosolic (pink) and TM (red). Based on the structural analysis reported in this study, each structure is labeled as inhibited or not inhibited. B, time-dependent RMSD evolution of the TM helix of PLB in the Ca²⁺-bound SERCA–PLB complexes. The RMSD was calculated using backbone alignment for TM helices of the SERCA. C, Cα RMSF values of PLB calculated from each independent MD trajectory. The shaded areas show the location of the cytosolic and TM helices of PLB.

We measured time-dependent root mean square deviation (RMSD) to determine the extent to which the position of the TM domain of PLB changes in the trajectories of the Ca²⁺-bound complexes. RMSD plots revealed that the position of PLB in the binding groove in all six trajectories does not deviate substantially (e.g. RMSD < 2.5 Å) from that determined by X-ray crystallography (Fig. 7B). In addition, root mean square fluctuation (RMSF) calculations using the main-chain Cα atoms show that the cytosolic helix of PLB is highly mobile in solution (Fig. 7C). This behavior corresponds to the inherent diffusion of the helical domain through the viscous bilayer surface, and it is uncorrelated with the presence or absence of inhibitory contacts. The RMSF values of the TM domain residues in all MD trajectories are substantially smaller than those calculated for the cytosolic helix; this indicates that the TM domain of PLB has very low mobility in the microsecond time scale. The RMSF values calculated for the TM domain of PLB, and particularly those around the residue Asn³⁴, are virtually identical both in the presence and absence of intermolecular inhibitory contacts (Fig. 7C).

We also calculated changes in the tilt angle of the TM helix of PLB to complement RMSD and RMSF measurements. We measured the relative tilt angle of the TM helix using the crystal structure of SERCA–PLB as a reference (Table 1). We found that the TM helix exhibits on average a 3.6° increase in the tilt angle relative to the position of PLB in the crystal structure of the complex (Table 1). We found no correlation between the loss of inhibitory contacts and the small change in tilt angle, which indicates that saturating Ca²⁺ conditions do not have an effect on the position of PLB in the complex. Hence, it is likely that the small changes in tilt angle are inherent to the PLB dynamics induced by the annular lipid shell surrounding the complex (44).

Relief of inhibitory contacts is not accompanied by substantial changes in the structure of PLB in the complex, so disruption of SERCA–PLB inhibitory contacts must occur locally at the interface of the complex. To test this hypothesis, we measured the fraction of native inhibitory contacts, Q_inh, between SERCA and PLB residues Leu³¹, Asn³⁴, Phe³⁵, and Ile³⁸; these residues, located near the cytosolic side of the complex, play important roles in inhibition of SERCA (45). Analysis of the Q_inh values showed that in the complexes with intact inhibitory contacts (CAL1 and CAL3), there is a high retention of native inhibitory contacts (Q_inh > 0.8) between key PLB residues and SERCA (Table 1). As anticipated, there is a substantial decrease in native inhibitory contacts (Q_inh = 0.52–0.62) for PLB residue Asn³⁴ in the trajectories where inhibitory contacts are disrupted (CAL2, CAL4, CAL5, and CAL6; Table 1). However, we found that a loss in native inhibitory contacts also occurs, albeit more moderately, at PLB positions Leu³¹, Phe³⁵, and Ile³⁸ (Table 1). This indicates that saturating Ca²⁺ conditions primarily affect local intermolecular interactions involving the side chain of PLB residue Asn³⁴, but also affect intermolecular interactions involving PLB residues within the inhibitory site at the SERCA–PLB interface.

Structure of the transport sites upon Ca²⁺-induced disruption of SERCA–PLB inhibitory interactions

We determined whether the structural changes following disruption of inhibitory SERCA–PLB interactions correspond to those associated with the formation of competent transport sites. To this aim, we measured the RMSD values for residues in sites I and II between the MD trajectories and the crystal structure of SERCA bound to two Ca²⁺ ions (E1·2Ca²⁺, PDB entry 1SU4). We also performed distance measurements to determine whether the location of the Ca²⁺ in the MD trajectories corresponds to that determined by X-ray crystallography (25).

Visualization of the transport sites in the trajectories CAL1 and CAL3, where the inhibitory SERCA–PLB interactions are intact, show that sites I and II are collapsed (Fig. 8). Calculated RMSD values for sites I and II (RMSD >2.7 Å) indicate that there is a poor overlap between these trajectories and the crystal structure of E1·2Ca²⁺ (Table 2). In these trajectories, the position of Ca²⁺ does not overlap with any of the two sites resolved by X-ray crystallography (Fig. 8). Instead, the Ca²⁺ ion binds to a location that is distant from sites I (r ≈ 5.5 Å) and site II (r ≈ 3.5 Å). These measurements indicate that in the presence of inhibitory SERCA–PLB contacts, Ca²⁺ does not bind to either site I or II, and the residues in the transport sites adopt a noncompetent structure.

Figure 8.

Ca²⁺ interactions with the transport sites of SERCA in the SERCA–PLB complex. Representative structures extracted from each MD simulation illustrate the location of Ca²⁺ (yellow sphere) and the structural arrangement of the residues that occlude Ca²⁺ in the transport sites (orange sticks). For comparison, each MD structure was superposed on the crystal structure of the Ca²⁺-bound SERCA, E1·2Ca²⁺ (25), to show the structure and location of the competent transport sites I and II; the side chains and Ca²⁺ ions resolved in the crystal structure are shown as blue sticks and cyan spheres, respectively. Based on the structural characteristics of each configuration, the structures of the transport sites are labeled as inhibited or not inhibited.

Table 2.

Structural features of the transport sites at saturating Ca²⁺ conditions

Trajectory	RMSD of heavy atomsa,b		Distance, ra,c
	Site I	Site II	Ca2+-site I	Ca2+-site II
	Å		Å
CAL1	2.8 ± 0.2	2.7 ± 0.3	5.5 ± 0.9	3.1 ± 0.5
CAL2	2.2 ± 0.3	4.4 ± 0.5	1.9 ± 0.2	4.2 ± 0.2
CAL3	3.2 ± 0.1	4.6 ± 0.2	5.6 ± 0.6	4.0 ± 0.4
CAL4	2.1 ± 0.1	4.7 ± 0.3	2.2 ± 0.2	4.2 ± 0.3
CAL5	1.7 ± 0.1	3.8 ± 0.3	2.2 ± 0.3	3.8 ± 0.3
CAL6	2.0 ± 0.1	4.9 ± 0.5	2.1 ± 0.3	4.0 ± 0.2

^a Values reported as average ± S.D.

^b RMSD was calculated using heavy atom alignment, with E1·2Ca²⁺ crystal structure (PDB code 1SU4 (25)) as reference.

^c Distances relative to the position of Ca²⁺ in sites I and II determined by x-ray crystallography (25).

In the MD simulations where the inhibitory SERCA–PLB are disrupted (trajectories CAL2, CAL4, CAL5, and CAL6), the structure of transport site II also deviates substantially from that in the crystal structure of E1·2Ca²⁺ (Fig. 8 and Table 2). However, we found that Ca²⁺ ion binds in a reproducible manner to a location that partially overlaps that of site I (r ≈ 2 Å) determined by X-ray crystallography (Fig. 8 and Table 2). When a single Ca²⁺ ion binds to this site, residues Glu⁷⁷¹, Thr⁷⁹⁹, Asp⁸⁰⁰, and Glu⁹⁰⁸ of site I adopt a structural arrangement that is similar to that found in the crystal structure of the PLB-free E1·2Ca²⁺ state of SERCA (RMSD <2.2 Å; Fig. 8). The structural rearrangements in the transport sites that follow Ca²⁺-induced relief of SERCA inhibition are reproducible in trajectories CAL2 and CAL4–CAL6 (Fig. 8) and correspond to the formation of a competent site I.

Discussion

We present a mechanistic study of the SERCA–PLB regulatory interactions at saturating Ca²⁺ conditions, thus providing quantitative insight into fundamental processes of activation of Ca²⁺ transport in the heart. We show that in a solution containing 10 mm Ca²⁺, calcium ions interact primarily with both cytosolic and luminal sides of SERCA and the lipid headgroups. Here, we show for the first time (to our knowledge) that at superphysiological Ca²⁺ conditions, Ca²⁺ ions interact with the luminal C-terminal region of PLB, but not with the cytosolic domain of PLB or the cytosolic side of the SERCA–PLB interface. This indicates that Ca²⁺ does not compete with PLB at the interface of the complex and does not have a direct effect on the structural dynamics and stability of unphosphorylated PLB. Previous FRET spectroscopy experiments support our data and show that saturating Ca²⁺ conditions alter neither the structural dynamics of unphosphorylated PLB nor the stability of the SERCA–PLB heterodimer (11).

At [Ca²⁺] = 10 mm, Ca²⁺ ions interact with SERCA at the entrance of the pathway that connects the cytosol with the transport sites, in agreement with previous studies by our group showing that PLB does not block metal ion binding to this region of SERCA (16, 17). Recognition of Ca²⁺ by SERCA is facilitated primarily by residues Asp⁵⁹ and Glu³⁰⁹, in agreement with mutagenesis studies of the pump (46). We found that Glu³⁰⁹ translocates a single Ca²⁺ ion from Asp⁵⁹ to Asp⁸⁰⁰, a critical residue in the transport sites that coordinates Ca²⁺ at sites I and II (25). This mechanism for Ca²⁺ translocation is in qualitative agreement with Brownian dynamics studies showing that fast Ca²⁺ binding to the transport sites is primarily guided by Glu³⁰⁹ (47).

In the absence of adequate charge neutralization of the transmembrane transport sites, SERCA denaturalization occurs very rapidly even within the native membrane at physiological pH (48, 49). Previous studies have shown that this electric charge can be compensated in the absence of Ca²⁺ by transport site protonation (16, 17) or by binding of metal ion K⁺ (50), Na⁺ (51–53), or Mg²⁺ (54, 55). This suggests that superphysiological concentrations of Ca²⁺ used in this study simply satisfy transport site charge neutralization and that the Ca²⁺-bound state of the SERCA–PLB complex might not represent a functional state in the cell. However, only a single Ca²⁺ ion occupies the transport sites at a time despite the superphysiological Ca²⁺ concentrations used in this study. This finding is consistent with previous studies showing that the Ca²⁺ binding to SERCA occurs in a sequential manner (56–58) and indicates that the Ca²⁺-bound SERCA–PLB structure represents a functional state of the complex at saturating Ca²⁺ conditions. In the absence of other Ca²⁺ ions, a single Ca²⁺ bound to the SERCA–PLB complex produces a total [Ca²⁺] of ∼400 μm. This value falls in the middle of previous estimates at elevated cytosolic Ca²⁺ in the cardiomyocyte (59–61), so we used the Ca²⁺-bound SERCA–PLB complex as a starting structure to probe the structural mechanism for relief of SERCA inhibition by PLB.

In 30% of the MD trajectories, Ca²⁺ binds to Glu³⁰⁹ and Asp⁸⁰⁰ in an orientation that is similar to that initially found at the end of the 0.5-μs MD trajectory at [Ca²⁺] = 10 mm. In this configuration, the inhibitory contacts remain intact in the microsecond time scale, and transport sites I and II lack the competent structural organization that is distinctive of the Ca²⁺-bound state of SERCA (25, 53, 62). This structural arrangement corresponds to a Ca²⁺-bound, inhibited SERCA–PLB complex. Previous studies have shown that PLB binding decreases SERCA's apparent affinity for Ca²⁺ only by 2–3-fold in the micromolar range (63), whereas others have suggested that PLB suppresses Ca²⁺ binding to SERCA (64). Our simulations help reconcile these conflicting studies because they show that PLB-induced changes in the transport sites delay Ca²⁺ binding to either sites I or II, thus altering the apparent Ca²⁺ affinity of SERCA (34).

SERCA–PLB inhibitory interactions are disrupted in 70% of the MD trajectories of the Ca²⁺-bound complex. In these cases, the initially bound Ca²⁺ moves to site I and recruits the carboxylic groups of transport site residues Glu⁷⁷¹ and Asp⁸⁰⁰. These Ca²⁺-induced structural changes occur concomitantly with a loss in the intermolecular interaction between the side chain of PLB residue Asn³⁴ and the backbone oxygen of SERCA residue Gly⁸⁰¹. Our data indicates that this Ca²⁺-dependent relief inhibitory contacts does not result from a large structural rearrangements in the SERCA–PLB interface (22) or changes in the native structural dynamics of PLB in the complex. Instead, PLB remains bound to SERCA, but PLB residue Asn³⁴ becomes dynamically more disordered and is unable to establish inhibitory contacts with SERCA. FRET spectroscopy experiments support these findings and show that Ca²⁺ acts upon SERCA–PLB complex exclusively at the TM domain level and that unlike PLB phosphorylation, Ca²⁺ does not induce changes in the structural dynamics of PLB (11). Whereas these structural changes have not been observed directly by spectroscopy, the Ca²⁺-induced repositioning and mobility of PLB residue Asn³⁴ observed in our simulations have been seen in X-ray crystallography studies of the complex at [Ca²⁺] = 1 mm.³

What are the specific interactions between Ca²⁺ and the transport sites that induce relief of inhibition? The crystal structure of the complex between sarcolipin, a PLB analog, and SERCA revealed a single Mg²⁺ ion bound to transport site residues Glu⁷⁷¹ and Asp⁸⁰⁰ (55). In this structure, the intermolecular inhibitory interactions are partially altered, which suggests that binding of divalent metal ions in the transport sites is sufficient to reverse SERCA inhibition. However, extensive studies by our group have demonstrated that in the inhibitory complex, Mg²⁺ does not simultaneously interact with Asp⁸⁰⁰ and Glu⁷⁷¹ (17). Instead, Mg²⁺ adopts a rigid octahedral coordination geometry that has a preference for binding water molecules as opposed to bulky protein side chain dipoles (17). In addition, the ionic radius of Mg²⁺ is smaller than that of Ca²⁺ (65), so adding side chain dipoles to the coordination shell is thermodynamically more favorable for Ca²⁺ than for Mg²⁺, so Ca²⁺ can produce drier, bulkier coordination complexes (66, 67). This explains why Ca²⁺, but not Mg²⁺, recruits both Glu⁷⁷¹ and Asp⁸⁰⁰ in the transport sites (17, 54). Owing to these distinctive properties of Ca²⁺, the tug of war between the attraction of Glu⁷⁷¹ and Asp⁸⁰⁰ for Ca²⁺ drag the Gly⁸⁰¹ backbone along with them as they move in toward Ca²⁺. These Ca²⁺-induced changes destabilize the interaction between Gly⁸⁰¹ and PLB residue Asn³⁴ and induce relief of SERCA inhibition by PLB. It is our postulate that Glu⁷⁷¹ and Asp⁸⁰⁰ to a large degree define the range of coordination spheres that help preserve or disrupt the inhibitory contacts in the SERCA–PLB complex.

Finally, we asked whether the Ca²⁺-induced structural changes detected in our simulations produce an intermediate state in the pathway toward SERCA activation. First, we found that upon relief of inhibitory contacts, the side chain of Glu³⁰⁹ populates a geometry in which the carboxylic group points toward the cytosol. We propose that this orientation of Glu³⁰⁹ is essential for binding and gating of a second Ca²⁺ ion in the transport site II (68). Second, we found that relief of inhibitory SERCA–PLB interactions occurs only when a single Ca²⁺ binds near transport site I and in agreement with mutagenesis studies showing that binding of a single Ca²⁺ in the transport site I is sufficient to reverse SERCA inhibition by PLB (19). The Ca²⁺-induced structural rearrangements we detected in the simulations correspond to those associated with the formation of a competent transport site I and a vacant site II. This transport site preorganization facilitates binding of a second Ca²⁺ ion and subsequent Ca²⁺-induced activation of the pump (25, 50, 53, 62).

In summary, we demonstrate that at saturating Ca²⁺ concentrations, binding of a single Ca²⁺ ion shifts the equilibrium toward a noninhibited structure of the SERCA–PLB complex. Our findings indicate that Ca²⁺-induced reversal of SERCA inhibition depends solely on the ability of Ca²⁺ to diffuse into the transport sites and that the ability of Ca²⁺ to enter the transport sites is not influenced by PLB. Our findings also indicate that reversal of SERCA–PLB inhibition at saturating Ca²⁺ conditions is uncoupled from other regulatory mechanisms, such as the order-to-disorder structural transitions of PLB (12, 39–43). Therefore, the lack of a regulatory mechanism would explain the inability of saturating [Ca²⁺] to effectively reverse impaired SERCA-mediated Ca²⁺ transport (4, 70, 71) and maladaptations (72) characteristic of chronic heart failure.

Experimental procedures

Setting up SERCA–PLB at superphysiological concentrations of Ca²⁺

We used an atomic model of the full-length PLB bound to SERCA generated previously by our group (16) to simulate the inhibited SERCA–PLB complex at superphysiological Ca²⁺ conditions. We modeled transport site residues Glu³⁰⁹, Glu⁷⁷¹, and Asp⁸⁰⁰ as unprotonated and residue Glu⁹⁰⁸ as protonated. In addition, we adjusted the pK_a of other ionizable residues to a pH value of ∼7.2 using PROPKA version 3.1 (73, 74). The complex was inserted in a pre-equilibrated 120 × 120-Å bilayer of palmitoyl-2-oleoyl-sn-glycerol-phosphatidylcholine lipids. We used the first-layer phospholipids that surround SERCA in the E1 state (75) as a reference to insert the complex in the lipid bilayer. This initial system was solvated using TIP3P water molecules with a minimum margin of 15 Å between the protein and the edges of the periodic box in the z axis. Ca²⁺ and Cl⁻ ions were added to produce a CaCl₂ concentration of ∼10 mm required to match the experimental conditions previously used to obtain crystal structures of Ca²⁺-bound SERCA (24, 25).

Setting up the SERCA–PLB complex at saturating Ca²⁺ conditions

We used the structure of the complex bound to a single Ca²⁺ ion obtained at superphysiological [Ca²⁺] as a starting structure to simulate the SERCA–PLB complex at saturating Ca²⁺ conditions. We found that a single Ca²⁺ ion bound to the transport sites of SERCA corresponds to a total Ca²⁺ concentration of ∼400 μm. This total Ca²⁺ concentration is much higher than that estimated at rest (76) and is also in good agreement with previous estimates at elevated cytosolic Ca²⁺ (59–61). The SERCA–PLB-Ca²⁺-lipid complex was solvated using TIP3P water molecules. K⁺ and Cl⁻ ions were added to neutralize the system and to produce a KCl concentration of ∼100 mm.

Setting up the SERCA–PLB complex at free Ca²⁺ conditions

We used an atomic model of the full-length SERCA–PLB structure (16) to simulate the inhibited complex at free Ca²⁺ conditions. On the basis of our previous studies (16), we modeled transport site residues Glu³⁰⁹ and Asp⁸⁰⁰ as unprotonated and residues Glu⁷⁷¹ and Glu⁹⁰⁸ as protonated. The lipid–water–protein complex was prepared using the same protocol and KCl concentrations used for the complex at saturating Ca²⁺ conditions.

Molecular dynamics simulations

MD simulations of all systems were performed by using the program NAMD version 2.12 (77), with periodic boundary conditions (78), particle mesh Ewald (79, 80), a nonbonded cutoff of 9 Å, and a 2-fs time step. CHARMM36 force field topologies and parameters were used for the proteins (81), lipid (69), water, Ca²⁺, K⁺, and Cl⁻. The NPT ensemble was maintained with a Langevin thermostat (310 K) and an anisotropic Langevin piston barostat (1 atm). Fully solvated systems were first subjected to energy minimization and warmup for 200 ps. This procedure was followed by 10 ns of equilibration with backbone atoms harmonically restrained using a force constant of 10 kcal mol⁻¹ Å⁻². We performed one 0.5-μs MD simulation of SERCA–PLB at 10 mm Ca²⁺ and 12 independent 1-μs MD simulations: six of Ca²⁺-bound SERCA–PLB and six of SERCA–PLB in the absence of Ca²⁺.

Structural analysis and visualization

VMD (33) was used for analysis, visualization, and rendering of the structures. To visualize the Ca²⁺–protein and Ca²⁺–lipid interactions, we created a map of the weighted mass density of Ca²⁺ using a grid resolution of 1 Å and a cutoff distance of 3.5 Å between Ca²⁺ and the protein/lipid atoms. This is achieved by replacing each atom in the selection with a normalized Gaussian distribution of width equal to the atomic radius. The distributions are then additively distributed on a grid. The final map is calculated by computing the mass density of Ca²⁺ for each step in the trajectory and averaged over the entire simulation time.

We calculated the fraction of native inhibitory contacts (Q_inh) between PLB residues Leu³¹, Asn³⁴, Phe³⁵, and Ile³⁸ and SERCA to measure the effect of calcium binding on the stability of the SERCA–PLB interface. Q_inh is defined by a list of native contact pairs (i,j) in the crystal structure of the complex. All pairs of heavy atoms i and j belonging to residues Xi and Xj are in contact if the distance between i and j is <7 Å. Q_inh is expressed as a number between 1 and 0, and it is calculated as the total number of native contacts for a given time frame divided by the total number of contacts in the crystal structure of the complex (PDB code 4KYT (15)).

Author contributions

E. F.-d. G. formal analysis; E. F.-d. G. validation; E. F.-d. G. and L. M. E.-F. investigation; E. F.-d. G. writing-review and editing; L. M. E.-F. conceptualization; L. M. E.-F. supervision; L. M. E.-F. funding acquisition; L. M. E.-F. writing-original draft.